Targeted disruption of tyrosinase causes melanin reduction in Carassius auratus cuvieri and its hybrid progeny

摘要

The white crucian carp(Carassius auratus cuvieri,WCC) not only is one of the most economically important fish in Asia,characterized by strong reproductive ability and rapid growth rates,but also represents a good germplasm to produce hybrid progenies with heterosis.Gene knockout technique provides a safe and acceptant way for fish breeding.Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic studies and hybridization breeding.Tyrosinase(TYR) is a key enzyme in melanin synthesis.Depletion of tyr in zebrafish and mice results in mosaic pigmentation or total albinism.Here,we successfully used CRISPR-Cas9 to target tyr in WCC and its hybrid progeny(WR) derived from the cross of WCC(♀) and red crucian carp(Carassius auratus red var.,RCC,♂).The level of TYR protein was significantly reduced in mutant WCC.Both the mutant WCC and the mutant WR showed different degrees of melanin reduction compared with the wild-type sibling control fish,resulting from different mutation efficiency ranging from 60% to 90%.In addition,the transcriptional expression profiles of a series of pivotal pigment synthesis genes,i.e.tyrp1,mitfa,mitfb,dct and sox10,were down-regulated in tyr-CRISPR WCC,which ultimately caused a reduction in melanin synthesis.These results demonstrated that tyr plays a key role in melanin synthesis in WCC and WR,and CRISPR-Cas9 is an effective tool for modifying the genome of economical fish.Furthermore,the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formation in non-model fish.